Differential lipid analysis of oxaliplatin-sensitive and resistant HCT116 cells reveals different levels of drug-induced lipid droplet formation.

Lipid droplets (LDs) are intracellular storage vesicles composed of a neutral lipid core surrounded by a glycerophospholipid membrane. LD accumulation is associated with different stages of cancer progression and stress responses resulting from chemotherapy. In previous work, a novel dual nano-electrospray ionization source and data-dependent acquisition method for measuring the relative abundances of lipid species between two extracts were described and validated. Here, this same source and method were used to determine if oxaliplatin-sensitive and resistant cells undergo similar lipid profile changes, with the goal of identifying potential signatures that could predict the effectiveness of an oxaliplatin-containing treatment. Oxaliplatin is commonly used in the treatment of colorectal cancer. When compared to a no-drug control, oxaliplatin dosing caused significant increases in triglyceride (TG) and cholesterol ester (CE) species. These increases were more pronounced in the oxaliplatin-sensitive cells than in oxaliplatin-resistant cells. The increased neutral lipid abundance correlated with LD formation, as confirmed by confocal micrographs of Nile Red-stained cells. Untargeted proteomic analyses also support LD formation after oxaliplatin treatment, with an increased abundance of LD-associated proteins in both the sensitive and resistant cells.

Paper-Based Coculture Platform to Evaluate the Effects of Fibroblasts on Estrogen Signaling in ER+ Breast Cancers.

Cell-based assays enable molecular-level studies of cellular responses to drug candidates or potential toxins. Transactivation assays quantify the activation or inhibition of nuclear receptors, key transcriptional regulators of gene targets in mamalian cells. One such assay couples the expression of luciferase to the transcriptional activity of estrogen receptor-alpha (ERα). While this assay is regularly used to screen for agonists and antagonists of the estrogen signaling pathway, the setup relies on monolayer cultures in which cells are plated directly onto the surface of cell-compatible plasticware. The tumor microenvironment is more than a collection of cancerous cells and is profoundly influenced by tissue architecture, the presence of extracellular matrices, and intercellular signaling molecules produced by non-cancerous neighboring cells (e.g., fibroblasts). There exists a need for three-dimensional culture platforms that can be rapidly prototyped to assess new configurations and readily produced in the large numbers needed for translational studies and screening applications. Here, we demonstrate the utility of the paper-based culture platform to probe the effects of intercellular signaling between two cell types. We used paper scaffolds to generate tumor-like environments, forming a defined volume of breast cancer cells suspended in collagen. By placing the paper scaffolds in commercial 96-well plates, we compared monocultures of only breast cancer cells with coculture configurations containing fibroblasts in different locations that mimicked the stages of breast cancer progression. We show that ERα transactivation in the T47D-KBluc cell line is affected by the presence, number, and proximity of fibroblasts, and is a consequence of intercellular signaling molecules. After screening a small library of fibroblast-secreted signaling molecules, we showed that interleukin-6 (IL-6) was the primary driver of reduced estradiol sensitivity. These effects were mitigated in the coculture configurations by the addition of an IL-6 neutralizing antibody. We also assessed estrogen receptor expression and transcriptional regulation, further demonstrating the utility of the paper-based platform for detailed mechanistic studies.

DiffN Selection of Tandem Mass Spectrometry Precursors.

Current data-dependent acquisition (DDA) approaches select precursor ions for tandem mass spectrometry (MS/MS) characterization based on their absolute intensity, known as a TopN approach. Low-abundance species may not be identified as biomarkers in a TopN approach. Herein, a new DDA approach is proposed, DiffN, which uses the relative differential intensity of ions between two samples to selectively target species undergoing the largest fold changes for MS/MS. Using a dual nano-electrospray (nESI) ionization source which allows samples contained in separate capillaries to be analyzed in parallel, the DiffN approach was developed and validated with well-defined lipid extracts. A dual nESI source and DiffN DDA approach was applied to quantify the differences in lipid abundance between two colorectal cancer cell lines. The SW480 and SW620 lines represent a matched pair from the same patient: the SW480 cells from a primary tumor and the SW620 cells from a metastatic lesion. A comparison of TopN and DiffN DDA approaches on these cancer cell samples highlights the ability of DiffN to increase the likelihood of biomarker discovery and the decreased probability of TopN to efficiently select lipid species that undergo large fold changes. The ability of the DiffN approach to efficiently select precursor ions of interest makes it a strong candidate for lipidomic analyses. This DiffN DDA approach may also apply to other molecule classes (e.g., other metabolites or proteins) that are amenable to shotgun analyses.

Selecting the appropriate indirect viability assay for 3D paper-based cultures: a data-driven study.

Cellular viability measurements quantify decreased proliferation or increased cytotoxicity caused by drug candidates or potential environmental toxins. Direct viability measures count each cell to provide an accurate readout. This approach can prove analytically challenging and time-consuming when cells are maintained in 3D structures akin to tissues or solid tumors. While less labor-intensive, indirect viability measures can be less accurate due to the heterogeneous structural and chemical microenvironment that arises when cells are maintained in tissue-like architectures and in contact with extracellular matrices. Here we determine the analytical figures of merit of five indirect viability assays in the paper-based cell culture platform we continue to develop in our laboratory: calcein-AM staining, the CellTiter-Glo assay, imaging fluorescent protein expression, propidium iodide staining, and the resazurin assay. We also determined the compatibility of each indirect assay with hypoxic conditions, intra-experimental repeatability, inter-experimental reproducibility, and ability to predict a potency value for a known antineoplastic drug. Our results show that each assay has benefits and drawbacks to consider when choosing the appropriate readout to answer a particular research question. We also highlight that only one indirect readout is unaffected by hypoxia, a commonly overlooked variable in cell culture that likely yields inaccurate viability measures.

Evaluating the Impact of Physiologically Relevant Oxygen Tensions on Drug Metabolism in 3D Hepatocyte Cultures in Paper Scaffolds.

Oxygen is an essential regulator of cellular function and phenotype. Despite its importance, the incorporation of physiologically relevant oxygen tensions is often overlooked in experimental setups. Ambient oxygen tensions (pO2 ∼152 mmHg) are significantly higher than those in the alveolar-capillary barrier of the lung, which is the most oxygen-rich interface in the body (pO2 ∼104 mmHg). The discrepancy between standard culture practices and physiologically relevant oxygen tensions is more pronounced when considering the hepatocyte-lined sinusoids of the liver, whose pO2 values range from 65 mm Hg in the periportal region to 30 mm Hg in the perivenous region. Our previous work highlights the need to transition from standard culture conditions to more physiologically relevant microenvironments when predicting hepatocyte responses to drug candidates or potential toxins. This protocol details an experimental pipeline for quantifying differences in transcript levels, protein levels, and activity of the cytochrome P450 1A (CYP1A) enzyme family in hepatocytes maintained in a three-dimensional environment at ambient and physiologically relevant oxygen tensions. We quantify changes in transcript with qRT-PCR, protein expression with western blots, and activity with the ethoxyresorufin-O-deethylase (EROD) assay. This approach can be adapted to any drug-metabolizing enzyme. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Preparing tissue-like environments to evaluate HepG2 cells in paper-based cell culture platform at physiological oxygen levels Basic Protocol 2: Evaluating CYP1A activity of hepatocytes grown in the paper scaffolds using the EROD assay Basic Protocol 3: Evaluating CYP1A transcript levels of hepatocytes grown in the paper scaffolds using RT-qPCR Basic Protocol 4: Evaluating CYP1A protein levels of hepatocytes grown in the paper scaffolds using western blotting.

Synthesis and Surface Attachment of Molecular Re(I) Complexes Supported by Functionalized Bipyridyl Ligands.

Eleven 2,2'-bipyridine (bpy) ligands functionalized with attachment groups for covalent immobilization on silicon surfaces were prepared. Five of the ligands feature silatrane functional groups for attachment to metal oxide coatings on the silicon surfaces, while six contain either alkene or alkyne functional groups for attachment to hydrogen-terminated silicon surfaces. The bpy ligands were coordinated to Re(CO)5Cl to form complexes of the type Re(bpy)(CO)3Cl, which are related to known catalysts for CO2 reduction. Six of the new complexes were characterized using X-ray crystallography. As proof of principle, four molecular Re complexes were immobilized on either a thin layer of TiO2 on silicon or hydrogen-terminated silicon. The surface-immobilized complexes were characterized using X-ray photoelectron spectroscopy, IR spectroscopy, and cyclic voltammetry (CV) in the dark and for one representative example in the light. The CO stretching frequencies of the attached complexes were similar to those of the pure molecular complexes, but the CVs were less analogous. For two of the complexes, comparison of the electrocatalytic CO2 reduction performance showed lower CO Faradaic efficiencies for the immobilized complexes than the same complex in solution under similar conditions. In particular, a complex containing a silatrane linked to bpy with an amide linker showed poor catalytic performance and control experiments suggest that amide linkers in conjugation with a redox-active ligand are not stable under highly reducing conditions and alkyl linkers are more stable. A conclusion of this work is that understanding the behavior of molecular Re catalysts attached to semiconducting silicon is more complicated than related complexes, which have previously been immobilized on metallic electrodes.

HepaRG cells undergo increased levels of post-differentiation patterning in physiologic conditions when maintained as 3D cultures in paper-based scaffolds.

Monolayer cultures of hepatocytes lack many aspects of the liver sinusoid, including a tissue-level organization that results from extracellular matrix interactions and gradients of soluble molecules that span from the portal triad to the central vein. We measured the activity and transcript levels of drug-metabolizing enzymes in HepaRG cells maintained in three different culture configurations: as monolayers, seeded onto paper scaffolds that were pre-loaded with a collagen matrix, and when seeded directly into the paper scaffolds as a cell-laden gel. Drug metabolism was significantly decreased in the presence of the paper scaffolds compared to monolayer configurations when cells were exposed to standard culture conditions. Despite this decreased function, transcript levels suggest the cells undergo increased polarization and adopt a biliary-like character in the paper scaffolds, including the increased expression of transporter proteins (e.g., ABCB11 and SLOC1B1) and the KRT19 cholangiocyte marker. When exposed to representative periportal or perivenous culture conditions, we observed in vivo zonal-like patterns, including increased cytochrome P450 (CYP) activity and transcript levels in the perivenous condition. This increased CYP activity is more pronounced in the laden configuration, supporting the need to include multiple aspects of the liver microenvironment to observe the post-differentiation processing of hepatocytes.

HepaRG cells undergo increased levels of post-differentiation patterning in physiologic conditions when maintained as 3D cultures in paper-based scaffolds.

Monolayer cultures of hepatocytes lack many aspects of the liver sinusoid, including a tissue-level organization that results from extracellular matrix interactions and gradients of soluble molecules that span from the portal triad to the central vein. We measured the activity and transcript levels of drug-metabolizing enzymes in HepaRG cells maintained in three different culture configurations: as monolayers, seeded onto paper scaffolds that were pre-loaded with a collagen matrix, and when seeded directly into the paper scaffolds as a cell-laden gel. Drug metabolism was significantly decreased in the presence of the paper scaffolds compared to monolayer configurations when cells were exposed to standard culture conditions. Despite this decreased function, transcript levels suggest the cells undergo increased polarization and adopt a biliary-like character in the paper scaffolds, including the increased expression of transporter proteins (e.g., ABCB11 and SLOC1B1) and the KRT19 cholangiocyte marker. When exposed to representative periportal or perivenous culture conditions, we observed in vivo zonal-like patterns, including increased cytochrome P450 (CYP) activity and transcript levels in the perivenous condition. This increased CYP activity is more pronounced in the laden configuration, supporting the need to include multiple aspects of the liver microenvironment to observe the post-differentiation processing of hepatocytes.

Cellular Invasion Assay for the Real-Time Tracking of Individual Cells in Spheroid or Tumor-like Mimics.

Cellular invasion is the gateway to metastasis, with cells moving from a primary tumor into neighboring regions of healthy tissue. Invasion assays provide a tractable experimental platform to quantitatively assess cellular movement in the presence of potential chemokines or inhibitors. Many such assays involve cellular movement from high cell densities to cell-free regions. To improve the physiological relevance of such assays, we developed an assay format to track cellular movement throughout a uniform density of cells. This assay format imparts diffusion-dominated environments along the channel, resulting in oxygen and nutrient gradients found in spheroids or poorly vascularized tumors. By incorporating oxygen- and pH-sensing films, we quantified spatial and temporal changes in the extracellular environment while simultaneously tracking the movement of a subset of cells engineered to express fluorescent proteins constitutively. Our results show the successful invasion into neighboring tissues likely arises from a small population with a highly invasive phenotype. These highly invasive cells continued to move throughout the 48 h experiment, suggesting they have stem-like or persister properties. Surprisingly, the distance these persister cells invaded was unaffected by the density of cells in the channel or the presence or absence of an oxygen gradient. While these datasets cannot determine if the invasive cells are inherent to the population or if diffusion-dominated environments promote them, they highlight the need for further study.

HepaRG cells adopt zonal-like drug-metabolizing phenotypes under physiologically relevant oxygen tensions and Wnt/ß-catenin signaling.

The cellular microenvironment plays an important role in liver zonation, the spatial distribution of metabolic tasks amongst hepatocytes lining the sinusoid. Standard tissue culture practices provide an excess of oxygen and a lack of signaling molecules typically found in the liver. We hypothesized that incorporating physiologically relevant environments would promote post-differentiation patterning of hepatocytes and result in zonal-like characteristics. To test this hypothesis, we evaluated the transcriptional regulation and activity of drug-metabolizing enzymes in HepaRG cells exposed to three different oxygen tensions, in the presence or absence of Wnt/ß-catenin signaling. The drug-metabolizing activity of cells exposed to representative periportal (11% O2) or perivenous (5% O2) oxygen tensions were significantly less than cells exposed to ambient oxygen. A comparison of cytochrome P450 (CYP) 1A2, 2D6, and 3A4 activity at PP and PV oxygen tensions showed significant increases at the lower oxygen tension. The activation of the Wnt/ß-catenin pathway only modestly impacted CYP activity at PV oxygen tension, despite a significant increase in CYP expression under this condition. Our results suggest oxygen tension is the major contributor to zonal patterning in HepaRG cells, with the Wnt/ß-catenin signaling pathway playing a lesser albeit important role. Our datasets also highlight the importance of including activity-based assays, as transcript data alone does not provide an accurate picture of metabolic competence. Significance Statement This work investigates the post-differentiation patterning of HepaRG cells cultured at physiologically relevant oxygen tensions, in the presence and absence of Wnt/ß-catenin signaling. HepaRG cells exposed to periportal (11% O2) or perivenous (5% O2) oxygen tensions display zonation-like patterning of both cytochrome P450 (CYP) and glucuronosyltransferase (UGT) enzymes. These datasets also suggest that oxygen is a primary regulator of post-differentiation patterning, with Wnt/ß-catenin having a lesser effect on activity but a significant effect on transcriptional regulation of these enzymes.

Spatially resolved quantification of drug metabolism and efficacy in 3D paper-based tumor mimics.

Paper-based cultures are an emerging platform for preparing three-dimensional (3D) tissue- and tumor-like structures. The ability to stack individual sheets of cell-containing paper affords a modular means of assembling structures with defined cellular compositions and microenvironments. These layered stacks are easily separated at the end of an experiment, providing spatially resolved populations of live cells for further analysis. Here we describe a workflow in which cell viability, drug penetration, and drug metabolism are quantified in a spatially resolved manner. Specifically, we mapped the distribution of the drug irinotecan and its bioactive metabolite SN38 in a colorectal cancer cell-containing stacked structure with liquid chromatography-mass spectrometry (LC-MS). This paper provides the first example of a 3D culture platform that quantifies viability and drug metabolism in a spatially resolved manner. Our data show that cells at the bottom of the stack are more drug-resistant than layers in contact with the culture medium, similar to cells in the nutrient-poor center of a proliferating tumor being more drug-resistant than the rapidly dividing cells at its periphery. The powerful combination of quantitative viability and drug metabolism measurements will enable future studies to determine the exact mechanism(s) of drug resistance in different regions of a tumor.

Physiologically relevant oxygen tensions differentially regulate hepatotoxic responses in HepG2 cells.

This study evaluates the impact of physiologically relevant oxygen tensions on the response of HepG2 cells to known inducers and hepatotoxic drugs. We compared transcriptional regulation and CYP1A activity after a 48 h exposure at atmospheric culture conditions (20% O2) with representative periportal (8% O2) and perivenous (3% O2) oxygen tensions. We evaluated cellular responses in 2D and 3D cultures at each oxygen tension in parallel, using monolayers and a paper-based culture platform that supports cells suspended in a collagen-rich environment. Our findings highlight that the toxicity, potency, and mechanism of action of drugs are dependent on both culture format and oxygen tension. HepG2 cells in 3D environments at physiologic oxygen tensions better matched primary human hepatocyte data than HepG2 cells cultured under standard conditions. Despite altered transcriptional regulation with decreasing oxygen tensions, we did not observe the zonation patterns of drug-metabolizing enzymes found in vivo. Our approach demonstrates that oxygen is an important regulator of liver function but it is not the sole regulator. It also highlights the utility of the 3D paper-based culture platform for continued mechanistic studies of microenvironmental influences on cellular responses.

Quantitative Effects of Disorder on Chemically Modified Amorphous Carbon Electrodes.

Real materials are disordered. This disorder influences the properties of these materials and the chemical processes that occur at their interfaces. Gaining a molecular-level understanding of the underlying physical manifestations caused by disordered materials is crucial to unraveling and ultimately controlling the efficiency and performance of these materials in a range of energy-related devices. This understanding necessitates measurement techniques through which disorder can be detected, quantified, and monitored. However, such quantitative measurements are notoriously difficult, as effects often average out in ensemble measurements. In this work, we describe how a combination of electrochemical and spatially resolved surface spectroscopy measurements illuminate a molecular-level picture of disorder in materials. Using amorphous carbon as an intrinsically disordered material, we covalently attached a monolayer of ferrocene. Interfacial electron transfer across the amorphous carbon-ferrocene interface is highly sensitive to disruptions of order. By systematically varying linker properties and surface loadings, the influence of lateral interactions between nonuniformly distributed ferrocene headgroups on ensemble electrochemical measurements is demonstrated. Electrochemical and imaging data collectively indicate that conformational flexibility of the ferrocene moieties provides a mechanism to elude repulsive and unbalanced lateral interactions, while rigid linkages provide direct information about the underlying disorder of the material. This study is the first of its kind to quantify and visualize molecular disorder and heterogeneity with an experimental model accessed through ensemble measurements.

Tracking the invasion of breast cancer cells in paper-based 3D cultures by OCT motility analysis.

3D paper-based cultures (PBCs) are easy-to-use and provide a biologically representative microenvironment. By stacking a sheet of cell-laden paper below sheets containing cell-free hydrogel, we form an assay capable of segmenting cells by the distance they invaded from the original cell-seeded layer. These invasion assays are limited to end-point analyses with fluorescence-based readouts due to the highly scattering nature of the paper scaffolds. Here we demonstrate that optical coherence tomography (OCT) can distinguish living cells from the surrounding extracellular matrix (ECM) or paper fibers based upon their intracellular motility amplitude (M). M is computed from fluctuation statistics of the sample, rejects shot noise, and is invariant to OCT signal attenuation. Using OCT motility analysis, we tracked the invasion of breast cancer cells over a 3-day period in 4-layer PBCs (160-300 µm thick) in situ. The cell population distributions determined with OCT are highly correlated with those obtained by fluorescence imaging, with an intraclass correlation coefficient (ICC) of 0.903. The ability of OCT motility analysis to visualize live cells and quantify cell distributions in PBC assays in situ and longitudinally provides a novel means for understanding how chemical gradients within the tumor microenvironment affect cellular invasion.

Generating linear oxygen gradients across 3D cell cultures with block-layered oxygen controlled chips (BLOCCs).

Oxygen is a transcriptional regulator responsible for tissue homeostasis and maintenance. Studies relating cellular phenotype with oxygen tension often use hypoxia chambers, which expose cells to a single, static oxygen tension. Despite their ease of use, these chambers are unable to replicate the oxygen gradients found in healthy and diseased tissues. Microfabricated devices capable of imposing an oxygen gradient across tissue-like structures are a promising tool for these studies, as they can provide a high density of information in a single experimental setup. We describe the fabrication and characterization of a modular device, which leverages the gas-permeability of silicone to impose gradients of oxygen across cell-containing regions, assembled by layering sheets of laser cut acrylic and silicone rubber. The silicone also acts as a barrier, separating the flowing gases from the cell culture medium, preventing evaporation or bubble formation in experiments that require prolonged periods of incubation. The acrylic components provide a rigid framework to provide a sterile culture environment. Using oxygen-sensing films, we show the device can support gradients of different ranges and steepness by simply changing the composition of the gases flowing through the silicone components of the BLOCC. Using a cell-based reporter assay, we demonstrate that cellular responses to hypoxia are proportional to oxygen tension.

Developing a Drug Screening Platform: MALDI-Mass Spectrometry Imaging of Paper-Based Cultures.

Many potential chemotherapeutics fail to reach patients. One of the key reasons is that compounds are tested during the drug discovery stage in two-dimensional (2D) cell cultures, which are often unable to accurately model in vivo outcomes. Three-dimensional (3D) in vitro tumor models are more predictive of chemotherapeutic effectiveness than 2D cultures, and thus, their implementation during the drug screening stage has the potential to more accurately evaluate compounds earlier, saving both time and money. Paper-based cultures (PBCs) are an emerging 3D culture platform in which cells suspended in Matrigel are seeded into paper scaffolds and cultured to generate a tissue-like environment. In this study, we demonstrate the potential of matrix-assisted laser desorption/ionization-mass spectrometry imaging with PBCs (MALDI-MSI-PBC) as a drug screening platform. This method discriminated regions of the PBCs with and without cells and/or drugs, indicating that coupling PBCs with MALDI-MSI has the potential to develop rapid, large-scale, and parallel mass spectrometric drug screens.

Tissue Papers: Leveraging Paper-Based Microfluidics for the Next Generation of 3D Tissue Models.

Paper-based scaffolds support the three-dimensional culture of mammalian cells in tissue-like environments. These Tissue Papers, a name that highlights the use of materials obtained from (plant) tissue to generate newly functioning (human) tissue structures, are a promising analytical tool to quantify cellular responses in physiologically relevant extracellular gradients and coculture architectures. Here, we highlight current examples of Tissue Papers, commonly used methods of analysis, and current measurement challenges.

Hypoxia differentially regulates estrogen receptor alpha in 2D and 3D culture formats.

Hypoxia is a common feature in solid tumors. Clinical samples show a positive correlation between the expression of the hypoxia-inducible factor HIF-1α and estrogen receptor alpha (ERα) and a negative correlation between HIF-1α and hormone sensitivity. Results from monolayer cultures are in contention with clinical observations, showing that ER (+) cell lines no longer express ERα under hypoxic conditions (1% O2). Here, we compared the impact of hypoxia on the ERα signaling pathway for T47D cells in a 2D and 3D culture format. In the 2D format, the cells were cultured as monolayers. In the 3D format, paper-based scaffolds supported cells suspended in a collagen matrix. Using ELISA, Western blot, and immunofluorescence measurements, we show that hypoxia differentially regulates ERα protein levels in a culture environment-dependent manner. In the 2D format, the protein levels are significantly decreased in hypoxia. In the 3D format, the protein levels are maintained in hypoxia. Hypoxia reduced ERα transcriptional activation in both culture formats. These results highlight the importance of considering tissue dimensionality for in vitro studies. They also show that ERα protein levels in hypoxia are not an accurate indicator of ERα transcriptional activity, and confirm that a positive stain for ERα in a clinical sample may not necessarily indicate hormone sensitivity.